The phytohormone ethylene is a signaling molecule that regulates numerous physiological processes throughout the life cycle of plants, including responses during germination, flower and fruit development, as well as the response of the plants to a variety of environmental stressors, such as drought, heat, excessive salinity, and disease (see, e.g., Chen et al, 2005, Annals of Botany, 95:901-915; Czarny et al, 2006 Biotechnol. Adv., 24:410-419). Ethylene biosynthesis pathways and signaling/regulatory pathways and networks are well described. For example, see FIGS. 1 and 2 in Wang et al, “Ethylene Biosynthesis and Signaling Networks”, in The Plant Cell, 2002 (Eds. American Society of Plant Biologists) pages S131-S151.
Several key steps in the ethylene signal transduction pathway are highly regulated in plants. For both EIN2 (ETHYLENE-INSENSITIVE2) and EIN3 (ETHYLENE INSENSITIVE3) proteins, their expression is induced by ethylene, which leads to an increased ethylene response. In addition, both EIN2 and EIN3 proteins are targeted for turnover by ETP1 (EIN2 TARGETING PROTEIN1) and ETP2 (EIN2 TARGETING PROTEIN2) or EBF1 (EIN3-BINDING F-BOX PROTEIN1) and EBF2 (EIN3-BINDING F-BOX PROTEIN2) respectively. The turnover of these key response signaling proteins helps to maintain plants in a repressed or “off” state in the absence of the hormone ethylene. Constitutive overexpression of ETP1 or ETP2 (Qiao et al., Genes Dev., 2009 Feb. 15; 23(4):512-21 (published on-line Feb. 4, 2009) or EBF1 or EBF2 (Guo and Ecker, 2003 Cell, 115:667-677) in transgenic Arabidopsis resulted in partial ethylene-insensitivity and reduced accumulation of EIN2 or EIN3 protein respectively.
Commercially, a common way to regulate ethylene response in plants, including fruits and vegetables and flowers, involves the application of a chemical to the plant, fruit, flower or vegetable, such as, for example 1-methylcyclopropene (1-MCP; AgroFresh, Inc.). 1-MCP is a compound that is used as a plant growth regulator that prevents ethylene from attaching to its receptors in plant tissues. Its application thereby increases the ethylene insensitivity of the plant. The temporary ablation of ethylene sensitivity can increase the plants' resistance or tolerance to stress, delay ripening, senescence, or flowering, among other commercially valuable manipulations of plant growth.
More recently, proposals to transform plant cells genetically with modified ethylene response receptors or other proteins involved in the ethylene response in plants have been suggested, such as in e.g., U.S. Pat. No. 6,294,716; US Patent Application Publication Nos. 2006/0200875, 2005/0066389, 2005/0060772 and 2004/0128719, among others. Such systems are directed to expression of a variety of mutated genes in the ethylene pathways. These systems generally employ a variety of suggested promoters to drive expression of the proteins, including constitutive promoters and tissue-specific promoters.
While the use of chemically regulated gene expression systems have been proposed for use in plants generally (M. Padidim, 2003 Curr. Opin Plant Biol., 6(2):169-77), many such systems are experimental only, or have been reported to have certain disadvantages. Among these disadvantages are the use of toxic or volatile inducers, low induction levels, poor translocation/movement in the plant, a slow ability to “turn-off” the expression of the gene or insufficient specificity to an inducer that is non-toxic to plants, among other issues. Such gene expression systems are not universally useful in all plants and selection of the operable components and their assembly is often challenging.
In the examples of the prior art, expression of the ethylene pathway genes is typically always on in all tissues and parts of the plant or is always on in specific tissues of the plant. However, tissue-specific promoters or low level constitutive promoters can be leaky or induced by an undesirable inducer. Such conventional promoters do not permit tight regulation of hormonal expression in the plant. The timing, duration and level of expression of the ethylene pathway genes are critical for normal physiological function. The induction of ethylene insensitivity at will and for a determined period of time has not been successfully demonstrated by the prior art.
There remains a need in the art for compositions and methods that permit controllable temporal regulation of ethylene sensitivity. This is particularly important for a gene product the expression of which is directly induced by the presence of ethylene. Such compositions and methods are needed for safe and effective use in agricultural crops and foodstuffs, as well as in other plants.